Wright, Matthew

							                                                                                          Matthew Wright
                                                                                        ORCA Final Report

         Cationic Steroid Antibiotics (CSAs) are potent innovative antimicrobial agents. In particular, CSA-
13 has a very low minimum inhibitory concentration (MIC) against Pseudomonas aeruginosa at 2 µg/mL.
When dissolved in a solvent and combined with a commercial polymer, CSA-13 can be used to coat
endotracheal (ET) tubes. Biofilm formation on ET tubes can cause ventilator assisted pneumonia and is
potentially fatal. CSA-13-coated ET tubes are extremely effective in preventing biofilm formation. Testing
in standard Mueller-Hinton broth, we have been able to prevent P. aeruginosa growth on tubes for up to
28 days. This is compared to silver-coated endotracheal tubes (which are currently on the market) that
prevent biofilm for 1 day, and non-coated tubes that prevent no biofilm. In all our tube assays we
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inoculate with 10 bacteria every day.
         Both the Kimberly-Clark and Covidien companies have shown interest to potentially market CSA-
coated tubes. However, the companies require that the coated tubes work effectively under specified
conditions. This includes, the drug working against specified bacterial amounts in Trypticase Soy Broth
(TSB) which contains Bovine Serum albumin (BSA). Kimberly-Clark requires the ET assays to be run in
5% BSA solution while Covidien requires exposing the tubes to 30% BSA solution on day one and then
run in normal broth without BSA after that.
         In order for CSA-13 to optimally work under these conditions, there needs to be a constant flow of
at least 10 µg/mL eluting off the tubes. Initially high bursts of CSA-13 have been shown to come off the
tubes. The amount of CSA-13 that comes off lessens as time goes by, beginning with a high initial burst,
and then tapering off to 1-5 ug/mL. Our goal in our experiments was to control the amount of drug
coming off. Through this will hoped for prolonged anti-biofilm activity as well as quickening the timetable
for either Kimberly-Clark or Covidien to commercially market the drug.
         CSA-13 binds to polymers through ionic interactions. Since commercial polymers vary in
structure and charge, there is also variability with how well CSA-13 binds to certain polymers. In order to
control the flow of CSA-13 off of coated ET tubes we tested several ideas. A certain polymer labeled
“Polymer E” has a lot of ionic character and CSA-13 binds better to it than to other polymers, so we
began testing with it.
         The general procedure our assays were as follows: ET tubes were cut into 1 cm segments and
then dipped into the CSA-containing polymer solution. The target increase in weight for each segment
was 25mg. Each segment is allowed to dry and subsequently re-dipped until the target weight was
reached. These tubes were then tested in assays of inoculated TSB over several days. On key days, an
elution profile of the supernatant was taken. Through high-performance liquid chromatography & mass
spectroscopy, the amount of CSA-13 coming off into solution was quantified. Since the HPLC method is
time consuming and considering the breadth of our experiments, we did not take elution profiles everyday
to quantify the CSA eluting off every day. Therefore, some assays we tried to determine effectiveness
based on biofilm growth.
         One hypothesis we tested was whether autoclaving and lyophilizing the tubes in a cold vacuum
had an effect on setting the polymer. We ran an assay using the Covidien method against tubes that
were a 3:1 ratio of Polymer E and a 3.5% CSA solution. One group was then lyophilized only, and
another group was lyophilized and autoclaved. The group that was lyophilized only lasted 14 before
bacterial growth was detected. The group that was autoclaved and lyophilized, however, lasted 26 days
without bacterial growth. Tubes tested using the Kimberly-Clark method were likewise successful when
autoclaved. An assay ran using the same 3:1 Polymer E to CSA ratio lasted 21 days without bacterial
growth.
         Another approach tried was to add a topcoat the tubes. This simply included adding another
dipping of polymer solution that did not contain the drug. Through this we hoped to quell the initially high
bursts of CSA-13 which came off the tubes. ET tubes with topcoats of differing thicknesses were tested
in order to find a thickness that stabilizes the amount of CSA-13 that elutes off.
         We tested a group of tubes with a Tecoflex base, a new polymer which some collaborators had
sent to our lab asking us to test out. We tested a control group without a top coat, which consisted of a
1:1 ratio of Tecoflex to 3.5% CSA polymer solution. The corresponding group had a topcoat consisting of
a 10% Polymer E solution. We aimed for a 10mg top coat, while the initial coating was approximately
20mg. Both of these solutions were tested using the Kimberly-Clark method. These tubes were also
autoclaved and lyophilized. Results showed an initial burst of CSA-13 in both groups, averaging around
290 µg/mL. Subsequent days showed dramatic reduction in the amount of CSA eluting off of the tubes.
By day seven, the assay with the top coat was eluting 15 µg/mL CSA while the tubes without the topcoat
was eluting about 6 µg/mL CSA. By day fifteen, tubes with the topcoat were eluting about 3.5 µg/mL CSA
while the tubes without the topcoat were eluting less than 1 µg/mL.
          These findings were both positive and negative. The top-coated tubes clearly slow the elution of
CSA-13 from the surface of the tubes, thereby prolonging the time it takes biofilm to form (tubes with top
coats showed growth after 21 days, those without topcoats showed growth after 8 days). However, this
topcoat did not prevent the initial large burst of CSA-13 from the tubes.
          We also tested ET tubes that had coatings containing both CSA-13 and the compound EGTA.
CSA-13 and EGTA had shown some synergistic effects in grid assays where free-floating CSA-13 and
EGTA were tested against bacteria. An assay using a 3:1 Pol E/CSA-13 to EGTA solution was used to
coat tubes. Two assays were run, one in which the samples were autoclaved and lyophilized, and one
which was only lyophilized. Results were not as good as anticipated as both assays were able to
withstand inoculate for only 10 days. Elutions were therefore not taken.
          Several months into our work, one of our collaborators suggested that we try using silicone
polymers in which to embed CSA-13. Our drug is not soluble in silicone and thus our procedure for
making polymer solutions drastically changed. Instead of dissolving CSA-13, the hypothesis was to make
a suspension of CSA-13 in silicone, thereby making tiny little pockets of the drug within a hardened
silicone casing. CSA-13 from these pockets would seep out gradually and kill bacteria within proximity of
the tubes. In order to make a suspension with equally distributed CSA-13, we ordered a 50-micron sieve
through which we had to manually grind the drug. This made the CSA-13 particles fine enough to make
small embeddings throughout the silicone solution. Any larger particles would have made the drug
pockets too large and the drug would seep out too quickly to be effective in the long run. The brand of
liquid silicone we used came in two parts which had to me mixed in equal proportions to make the
silicone. We then added the varying amounts of CSA-13. ET tubes were then dipped once in the
solution and subsequently put into an oven for seven minutes to cure the silicone coating. These
samples were then put in a vacuum dryer and dried over night. Assays were then tested with and without
silicone-only top coats.
          Our tests with the silicone polymer proved to be so successful that we stopped all work with
Polymer E and began to work solely with silicone. Our Initial tests using 5 and 10% solutions of CSA-13
proved to not be enough CSA-13 to withstand even one day. Solutions of 33% were next tested only to
discover large amounts of CSA-13 precipitating off of the tubes. To combat this, we remade the samples
and added a topcoat of silicone. This method proved to have very positive results. The tubes were able
to kill bacterial inoculate for 21 days. Furthermore, CSA-13 elution was much more controlled. Days one
and two had elutions averaging 153 µg/mL with day three having an elution average of 68 µg/mL and day
four with 50 µg/mL. These results vary drastically with the solutions without the topcoats (those that
precipitated), which showed a day one burst of over 400 µg/mL while day two had a mere 30 µg/mL.
They were also significantly less than our assays run with Polymer E. Subsequent testing of other
percent solutions have been positive also. A 15% CSA-13 solution yielded a day one elution of just 50
µg/mL while days two and three were at 30 µg/mL. These assays lasted 18 days in the assay without
showing growth.
          With the success of the silicone assays, the lab continues to experiment with various percent
solutions as well as different top coats, including thinner topcoats that are diluted using hexanes as
solvent. Because of these successes, the work and data we spent on the Polymer E solutions is no
longer of value to us. However, the months we spent on it cannot be viewed as a waste of time and
resources since our failures led us and our collaborators to test other polymers like the silicone, which
have proved to be a big success.
          As initially speculated, the full breadth of our work on this project is continuing beyond my time
spent working in the lab. Presently, our associates at Kimberly-Clark have become more interested in
our work and have begun to work more closely with our lab. This was one of the big aims for working on
our experiments. In time, the company may decide to more fully invest in our work and eventually market
the drug commercially.